T cell inhibitory molecules such as CTLA-4, PD-1, and Tim-3 are important regulators of the T cell response that have emerged as valuable immunotherapeutic targets in cancer. Among T cell inhibitory molecules, Tim-3 is now recognized as a critical determinant of the dysfunctional or exhausted CD8+ T cells that arise in cancer. Indeed, we were the first to show that expression of Tim-3 specifically marks the most dysfunctional or exhausted population of CD8+ T cells present in tumor tissue and that immunotherapy that targets Tim-3 markedly improves the efficacy of immunotherapy that targets PD-1 for restoring function to exhausted CD8+ T cells and eliciting potent anti-tumor immunity. We have now discovered that expression of Tim-3 also marks a highly suppressive population of CD4+ FoxP3+ regulatory T cells (Treg) that is uniquely present in tumor tissue. Our preliminary data further indicate that Tim-3 promotes the suppressor function of Treg. Thus, Tim-3 can impact on tumor immunity through its actions in CD8+ T cells and Treg. However, how Tim-3 may function in CD8+ T cells to dampen their responses while in Treg function to promote their suppressive activity is not known. We have now discovered that TCF-1 (T cell factor 1) is specifically down-regulated in Tim-3+ CD8+ tumor- infiltrating lymphocytes (TILs) that exhibit dysfunctional phenotype. Our preliminary data further show that blockade of Tim-3 and PD-1 signaling, which we have shown abrogates T cell exhaustion, restores expression of TCF-1 in Tim-3+ CD8+ TILs. We have further found that TCF-1 is similarly down-regulated in Tim-3+ but not Tim-3- Treg and that Tim-3/PD-1 blockade restores TCF-1 expression in Tim-3+ Treg. TCF-1 is a key transcription factor in the canonical Wnt signaling pathway. TCF-1 is required for normal T cell development and for the generation of long-lived central memory CD8+ T cells but a role for Wnt signaling and TCF-1 in the development of T cell dysfunction/exhaustion has not been addressed. Interestingly, TCF-1 has recently been implicated in antagonizing regulatory T cell function, thus raising the possibility that Tim-3-drivn repression of TCF-1 underlies the potent suppressor function of Tim-3+ Treg. Based on our preliminary data, we hypothesize that Tim-3 suppresses the anti-tumor T cell response via a mechanism that involves repression of Wnt signaling and TCF-1 in intra-tumoral CD8+ T cells and Treg. We are in a unique position to dissect the role of Tim-3 and TCF-1 in determining the anti-tumor T cell response as we have available novel tools, including Tim- 3 and TCF-1 conditional knock-out mice as well as mice that selectively express activating versus repressive forms of TCF-1 specifically in lymphocytes. We will use these newly generated tools to define the mechanisms by which Tim-3 determines anti-tumor immunity and the role of TCF-1 in this process. We propose the following specific aims: 1) Define the role of Tim-3 in CD4+FoxP3+ regulatory T cells; and 2) Define the role of Wnt signaling/TCF-1 in Tim-3-driven suppression of anti-tumor immunity.